CN113200988B - Synthesis and application of bifunctional fluorescent probe for simultaneously detecting hydroxyl free radicals and viscosity - Google Patents

Abstract

其中，R＝氰基、乙酸乙酯基或苯并噻唑基。该类荧光探针利用羟基自由基芳香加成性质，在苯环上加成，随后与氰基发生关环，在430nm激发波长下发射500nm左右的强绿光。该类探针对羟基自由基的选择性高，反应后荧光产物具有水溶性较好，荧光量子产率高，斯托克斯位移大等优点。此外，氰乙烯单元可自由旋转，但当粘度增加时，旋转受阻，抑制了分子的非辐射损耗过程，在470nm激发波长下发射600nm以上的红光。此类探针可双通道同时检测羟基自由基和粘度，在分析化学、生命科学、生物传感等技术领域有着巨大的应用前景。The invention discloses a kind of bifunctional fluorescent probes for simultaneously detecting hydroxyl radicals and viscosity. The general chemical structure of the bifunctional fluorescent probes is as follows:

wherein R=cyano, ethyl acetate or benzothiazolyl. This kind of fluorescent probe utilizes the aromatic addition property of hydroxyl radical to add to the benzene ring, and then closes the ring with the cyano group, and emits a strong green light of about 500 nm at the excitation wavelength of 430 nm. Such probes have high selectivity to hydroxyl radicals, and the fluorescent products after the reaction have the advantages of good water solubility, high fluorescence quantum yield, and large Stokes shift. In addition, the vinyl cyanide unit can rotate freely, but when the viscosity increases, the rotation is hindered, suppressing the non-radiative depletion process of the molecule, emitting red light above 600 nm at the excitation wavelength of 470 nm. Such probes can simultaneously detect hydroxyl radicals and viscosity in two channels, and have great application prospects in analytical chemistry, life sciences, biosensing and other technical fields.

Description

Synthesis and Application of Bifunctional Fluorescent Probe for Simultaneous Detection of Hydroxyl Radical and Viscosity

technical field

The invention belongs to the technical field of analytical chemistry, and in particular relates to the synthesis and application of a bifunctional fluorescent probe capable of simultaneously detecting hydroxyl radicals and viscosity. Such probes rapidly and selectively detect hydroxyl radicals and viscosity from various reactive oxidizing species with a new mechanism, the green fluorescence channel selectively detects hydroxyl radicals, and the red fluorescence selective channel detects viscosity, with large Stokes It has the advantages of displacement, high fluorescence quantum yield, high detection sensitivity and visual detection.

Background technique

Reactive oxygen species include: hydroxyl radical (·OH), hypochlorous acid (HClO), peroxynitrite (ONOO ^- ), peroxy anion (O ₂ ^{· -} ), nitric oxide (NO), singlet oxygen ( ¹ O ₂ ) and hydrogen peroxide (H ₂ O ₂ ), which play important roles in different physiological and pathological processes (Nature, 2006, 443, 787-795; Nature Rev Immu. 2006, 6, 508-519). Among all reactive oxygen species, hydroxyl radicals are the most oxidatively active species, which have an extremely short lifespan and can react with many biomolecules such as DNA bases, lipids and proteins. Its overproduction causes cellular damage and is associated with various diseases. In addition, much evidence suggests that the production of ·OH and other reactive oxygen species can be used in cancer therapy (Nature, 2000, 407, 309–311). On the other hand, intracellular viscosity is a key factor controlling mass and signal transmission as well as interactions between biological macromolecules. In fact, it has been shown to be an important contributor or indicator of atherosclerosis, diabetes, Alzheimer's disease, and even cellular malignancies (Biochem. J., 1994, 298, 443-450; Psychopharmacology, 1999, 145, 175- 180), as it affects protein-protein interactions in the cell membrane. Therefore, monitoring intracellular OH levels and viscosity in cells is crucial for understanding its biological function.

Recently, several fluorescent probes for detecting OH and viscosity have been reported. Among them, fluorescent probes based on characteristic reactions of OH, such as aromatic addition, spin trapping and hydrogen abstraction, exhibit relatively good selectivity to other reactive oxygen species. For example, weakly fluorescent coumarin-3-carboxylic acid probes react with OH to generate fluorescent 7-hydroxy-coumarin-3-carboxylic acid derivatives through aromatic addition reactions (Anal. Sci., 2008, 24, 293 – 296); also developed spin trapping methods for nitroxyl radical probes with OH in DMSO (Org. Lett., 2012, 14, 50–53); Needle oxidation reacts to the corresponding cyanine dyes for the detection of _· OH and H2O2 ₍ Angew. Chem., Int. Ed., 2009, 48, 299-303). At present, a large number of fluorescent probes for detecting viscosity changes have also been reported. By inhibiting the rotation of the C=C double bond in the molecule and reducing the non-radiative loss of the probe molecule, high-sensitivity detection of viscosity can be achieved (Chem. Eur. J., 2021 , DOI: 10.1002/chem.202004888). There are few bifunctional probes that simultaneously detect hydroxyl radicals and viscosity changes. Therefore, it is still a great challenge to use a single fluorescent probe to achieve dual-channel discrimination and detection of hydroxyl radicals and viscosity simultaneously.

SUMMARY OF THE INVENTION

In view of the above situation, and overcoming some deficiencies of the prior art, the purpose of the present invention is to provide a kind of bifunctional fluorescent probes for simultaneously detecting hydroxyl radicals and viscosity. This type can rapidly and selectively detect hydroxyl radicals from various active oxidizing substances under specific detection conditions, and has a good fluorescence response to viscosity. The present invention also aims to provide a method for synthesizing and applying the above-mentioned fluorescent molecular probe with simple preparation method, high sensitivity, low detection limit and low cost.

The specific technical solution adopted by the present invention to solve the problem is to synthesize and prepare a bifunctional fluorescent probe that simultaneously detects hydroxyl radicals and viscosity, quantitatively analyze hydroxyl radicals and viscosity in the environment, and simultaneously distinguish and fluorescently image hydroxyl radicals in living cells. And the application of the device of viscosity, the general chemical structure of its bifunctional probe is as follows:

其中，R＝氰基、乙酸乙酯基或苯并噻唑基。

wherein R=cyano, ethyl acetate or benzothiazolyl.

Synthesis of a bifunctional fluorescent probe for simultaneous detection of hydroxyl radicals and viscosity, characterized in that the preparation method of the bifunctional fluorescent probe comprises the following steps:

Step 1. Synthesis of methyl 3-(8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]quinoxalin-5(1H)-yl)propanoate

a. Add an appropriate amount of 3-fluoro-4-nitroanisole and L-proline methyl ester hydrochloride into anhydrous acetonitrile, then add an appropriate amount of triethylamine and heat under reflux for 6 hours, filter and remove the triethylamine hydrochloride, spin dry solution,

b. Dissolve the spin-dried solution with methanol, slowly add an appropriate amount of ammonium chloride and zinc powder, stir at room temperature overnight, then filter to remove the zinc powder, spin-dry the solvent, add the obtained solid to water, filter and dry to obtain a green solid,

c. Dissolve the green solid with anhydrous tetrahydrofuran, add an appropriate amount of sodium borohydride and boron trifluoride ether, react at 90°C for 12 hours, after the reaction is complete, slowly pour into water, adjust the pH of the solution to 12 with sodium hydroxide, filter , the filtrate was extracted with ethyl acetate, the organic layer was spin-dried,

d. The above product was added to an appropriate amount of methyl acrylate and glacial acetic acid for reflux reaction for 6 hours, filtered to obtain 3-(8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2-a] Methyl quinoxalin-5(1H)-yl)propanoate;

Step 2. Synthesis of methyl 3-(7-formyl-8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2a]quinoxalin-5(1H)-yl)propanoate

1. Under nitrogen protection, slowly add an appropriate amount of dry and re-distilled N,N-dimethylformamide (DMF) to an equal volume of phosphorus oxychloride (POCl ₃ ), and stir at 20-50 ° C for 30- After 60 minutes, a yellow solution was obtained, and methyl 3-(8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]quinoxalin-5(1H)-yl)propanoate was The ester was dissolved in an appropriate amount of DMF, added dropwise to the yellow mixed solution, and the mixture continued to stir and react at 60°C for 12 hours under nitrogen protection; after the reaction was completed, the reaction solution was poured into an appropriate amount of ice water and adjusted with 20% NaOH solution The pH was adjusted to 5-6, then extracted with ethyl acetate, and the organic layer was spin-dried to obtain a yellow solution;

Step 3. Synthesis of 3-(7-(2,2-dicyanovinyl)-8-methoxy-2,3,3a,4tetrahydropyrrolo[1,2-a]quinoxaline-5 (1H)-yl) methyl propionate (take R=cyano as an example)

i. Methyl 3-(7-formyl-8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2a]quinoxalin-5(1H)-yl)propanoate and Malononitrile was added to an appropriate amount of ethanol, followed by an appropriate amount of piperidine, reacted overnight at room temperature, filtered, and the solid was vacuum-dried to obtain the bifunctional fluorescent molecular probe 3-(7-(2,2-dicyanovinyl) -8-Methoxy-2,3,3a,4tetrahydropyrrolo[1,2-a]quinoxalin-5(1H)-yl)propanoate methyl ester.

A method of using a bifunctional fluorescent probe for simultaneous detection of hydroxyl radicals and viscosity of the present invention: no special instructions, usually at room temperature, the probe molecules are dissolved in a phosphate buffer solution (PBS) whose aqueous phase is pH=7.4 It is used for the analysis and detection of hydroxyl radicals in the environment of the aqueous solution of the analyte and the analyte; when detecting the viscosity, the probe molecules are dissolved in methanol-glycerol solutions of different viscosities to detect the viscosity.

The specific features of the dual-function fluorescent probe of the present invention for simultaneous detection of hydroxyl radicals and viscosity are as follows: the dual-function fluorescent probe is dissolved in dimethyl sulfoxide (DMSO), the probe is dissolved in different gradients of viscosity, and the probe is directly excited at 470 nm. It emits red fluorescence above 600nm at a wavelength; the probe is dissolved in phosphate buffer solution (PBS) and reacted with hydroxyl radicals at room temperature for 15 minutes, and emits strong green fluorescence at about 500nm at an excitation wavelength of 430nm. Specific excitation and fluorescence emission signals are thus achieved to detect specific analytes. The above fluorescent molecular probes can simultaneously distinguish and detect hydroxyl radicals and viscosity under different detection conditions, and have no obvious response to other reactive oxygen species, active sulfur, common amino acids, metal ions and reactive nitrogen, and the detection limit of hydroxyl radicals As low as 183.3nM, high sensitivity to viscosity changes. Therefore, the dual-function fluorescent probe disclosed in the present invention can realize highly sensitive detection of both.

Description of drawings

Fig. 1 is the hydrogen nuclear magnetic resonance spectrum (R=cyano group) of the bifunctional fluorescent probe of the present invention.

Fig. 2 Fluorescence spectrum diagram (R = ethyl acetate) of the bifunctional fluorescent probe of the present invention to detect hydroxyl radicals and viscosity.

FIG. 3 is a fluorescence imaging diagram of simultaneous imaging of intracellular hydroxyl radicals and viscosity with the dual-functional fluorescent probe of the present invention (R=benzothiazolyl).

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings.

The synthetic route of the bifunctional fluorescent probe of the present invention is as follows:

Example 1. Synthesis of methyl 3-(8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]quinoxalin-5(1H)-yl)propanoate

a. Add 30g (175.31mmol) of 3-fluoro-4-nitroanisole and 37.5g (227.9mmol) of L-proline methyl ester hydrochloride to 200mL of anhydrous acetonitrile, and then add 73mL (525.9mmol) Triethylamine was heated under reflux for 6 hours, filtered to remove triethylamine hydrochloride, and the solution was spin-dried.

b. Dissolve the spin-dried solution with 300 mL of methanol, slowly add 46.76 g (874.13 mmol) of ammonium chloride and 57.15 g (874.13 mmol) of zinc powder, stir at room temperature overnight, then filter to remove the zinc powder, spin dry the solvent, and the solid Add water, filter and dry to obtain green solid,

c. Dissolve the green solid with 400 mL of anhydrous tetrahydrofuran, add 36.4 g (962.17 mmol) of sodium borohydride and 122 mL (962.17 mmol) of boron trifluoride ether, react at 90 ° C for 12 hours, after the reaction is complete, slowly pour into water, Adjust pH to 12 with sodium hydroxide, filter, extract the filtrate with ethyl acetate, spin dry the organic layer,

d. The above product was added to an appropriate amount of methyl acrylate for reflux reaction for 12 hours, filtered to obtain 3-(8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]quinoxaline Methyl -5(1H)-yl)propionate 26.4 g, yield 64.27%.

Example 2. Synthesis of methyl 3-(7-formyl-8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2a]quinoxalin-5(1H)-yl)propanoate ester

1. Under nitrogen protection, slowly add 10 mL (77.48 mmol) of phosphorous oxychloride (POCl 3 ) to 10 mL (77.48 mmol) of phosphorus oxychloride (POCl ₃ ) by adding an appropriate amount of dry and re-distilled 10 mL of N,N-dimethylformamide (DMF) at 20-50° C. Stir for 30-60 minutes to obtain a yellow solution, 10 g (43.04 mmol) of 3-(8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]quinoxaline-5( 1H)-yl) methyl propionate was dissolved in 40 mL of N,N-dimethylformamide (DMF), added dropwise to the above yellow mixed solution, and the mixture continued to stir and react at 60°C for 12 hours under nitrogen protection; the reaction After completion, the reaction solution was poured into an appropriate amount of ice water, and the pH was adjusted to 5-6 with 20% NaOH solution, and then a large amount of solid was precipitated, which was filtered to obtain 10.2 g of a yellow solid with a yield of 93.03%.

Example 3. Synthesis of 3-(7-(2,2-dicyanovinyl)-8-methoxy-2,3,3a,4tetrahydropyrrolo[1,2-a]quinoxaline- 5(1H)-yl) methyl propionate (R=cyano as an example)

i. Methyl 3-(7-formyl-8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2a]quinoxalin-5(1H)-yl)propanoate 200 mg (628.2 μmol) solid and 49.80 mg (753.8 μmol) malononitrile were added to 8 mL of ethanol, reacted overnight at room temperature, and filtered to obtain the fluorescent molecular probe 3-(7-(2,2-dicyanovinyl)- 8-Methoxy-2,3,3a,4tetrahydropyrrolo[1,2-a]quinoxalin-5(1H)-yl)propionic acid methyl ester 185 mg, yield 80.37%.

Example 4. Application of bifunctional fluorescent probes in the detection of hydroxyl radicals and viscosity in vitro (R=ethyl acetate)

The spectroscopic property experiment of the bifunctional fluorescent probe in the detection of hydroxyl radicals and viscosity of the present invention: the probe is dissolved in dimethyl sulfoxide (DMSO) to prepare a probe solution with a concentration of 1 mM, and the concentration is respectively 10 mM aqueous solution of hydroxyl radicals. The specific test method is as follows: take 20 μL of 1 mM probe solution, add an appropriate amount of analyte solution, and finally add an appropriate amount of phosphate buffer solution (PBS) (the total volume of each test sample is 2 mL). For example, when it is required to test the fluorescence intensity of the probe reacting with hydroxyl radicals when the hydroxyl radical concentration is 20 μM, the sample preparation is as follows: take 20 μL of 1 mM probe solution, 20 μL of 10 mM hydroxyl radical aqueous solution and 1960 μL of PBS buffer solution In a 2mL sample tube, shake it at room temperature for 15 minutes, and then use the excitation wavelength of 430nm to measure the fluorescence emission intensity. The specific test method for detecting the viscosity is basically the same. The preparation method is to take 20μL of 1mM probe solution and add 1960μL of different viscosity In solution, other test operations are similar to the above steps. The dual-function probe molecule realizes the simultaneous detection of hydroxyl radicals and viscosity with different excitation wavelengths and fluorescence emission signals, and has high sensitivity. The detection limit of hydroxyl radicals is as low as 183.3nM, and it is sensitive to viscosity changes. Imaging/quantitative analysis of endogenous hydroxyl radicals and viscosity changes in living cells.

Example 5. Dual-channel fluorescence imaging analysis of endogenous hydroxyl radicals and viscosity in RAW246.7 (macrophage) cells

RAW246.7 cells were passaged into confocal cell culture medium, and after culturing under standard growth conditions for 24 hours, an appropriate amount of probe (5 μM, R = benzothiazolyl) was added to continue to incubate under standard growth conditions for 30 minutes, and then Photographs were taken under a confocal fluorescence microscope, and the green and red fluorescence channels were used for fluorescence imaging of endogenous hydroxyl radicals and viscosity in RAW246.7 cells. As can be seen from Figure 3, the dual-functional fluorescent probe of the present invention successfully achieved cell Dual-Channel Fluorescence Imaging Analysis of Endogenous Hydroxyl Radicals and Viscosity.

The dual-function fluorescent probe for simultaneously detecting hydroxyl radicals and viscosity provided by the present invention utilizes the aromatic addition property of hydroxyl radicals, and then undergoes a cyclization reaction with cyano groups, and can rapidly respond to hydroxyl radicals with high selectivity. Under the excitation wavelength of 430nm, it emits green light of about 500nm; the free-rotating vinyl cyanide moiety is suppressed by the viscosity change, thereby suppressing the non-radiative loss process of the probe molecule, realizing real-time monitoring of the viscosity change, and emitting light above 600nm under the excitation wavelength of 470nm. Red light, the fluorescence phenomenon is obvious. Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be construed as limiting the present invention. Various modifications and alternatives to the present invention will be apparent to those skilled in the art upon reading the foregoing. Therefore, the application of the bifunctional fluorescent probe with the technical features described in this paper to detect hydroxyl radicals and viscosity at the same time falls within the protection scope of this patent.

Claims (4)

1. The difunctional fluorescent probe for simultaneously detecting the hydroxyl free radicals and the viscosity is characterized by having the following structural general formula:

wherein R is cyano, ethyl acetate or benzothiazolyl.

2. The synthesis of a bifunctional fluorescent probe according to claim 1, characterized in that the synthesis method of the bifunctional fluorescent probe comprises the following steps:

step 1 Synthesis of methyl 3- (8-methoxy-2, 3,3a, 4-tetrahydropyrrolo [1,2-a ] quinoxalin-5 (1H) -yl) propionate

a. Adding appropriate amount of 3-fluoro-4-nitrobenzyl ether and L-proline methyl ester hydrochloride into anhydrous acetonitrile, adding appropriate amount of triethylamine, heating, refluxing for 6 hr, filtering to remove triethylamine hydrochloride, spin drying the solution,

b. dissolving the dried solution with methanol, slowly adding appropriate amount of ammonium chloride and zinc powder, stirring at room temperature overnight, filtering to remove zinc powder, spin drying the solvent, adding the obtained solid into water, filtering and drying to obtain green solid,

c. dissolving the green solid with anhydrous tetrahydrofuran, adding appropriate amount of sodium borohydride and boron trifluoride diethyl etherate, reacting at 90 deg.C for 12 hr, slowly pouring into water after reaction is completed, adjusting pH to 12 with sodium hydroxide, filtering, extracting the filtrate with ethyl acetate, spin drying the organic layer,

d. adding the product into a proper amount of methyl acrylate and glacial acetic acid for reflux reaction for 6 hours, and filtering to obtain 3- (8-methoxy-2, 3,3a, 4-tetrahydropyrrolo [1,2-a ] quinoxaline-5 (1H) -yl) methyl propionate;

step 2 Synthesis of methyl 3- (7-formyl-8-methoxy-2, 3,3a, 4-tetrahydropyrrolo [1,2a ] quinoxalin-5 (1H) -yl) propionate

I. Under the protection of nitrogen, adding a proper amount of dry redistilled N, N-Dimethylformamide (DMF) slowly into equal volume of phosphorus oxychloride (POCl)₃) Stirring at 20-50 deg.C for 30-60 min to obtain yellow solution, and mixing 3- (8-methoxy-2, 3,3a, 4-tetrahydropyrrolo [1,2-a ]]Dissolving quinoxaline-5 (1H) -yl) methyl propionate in a proper amount of DMF, dropwise adding the solution into a yellow mixed solution, and continuously stirring the mixture at 60 ℃ under the protection of nitrogen for reacting for 12 hours; after the reaction is completed, pouring the reaction solution into a proper amount of ice water, adjusting the pH to 5-6 by using a 20% NaOH solution, extracting by using ethyl acetate, and spin-drying an organic layer to obtain a yellow solution;

step 3. Synthesis of methyl 3- (7- (2, 2-dicyanovinyl) -8-methoxy-2, 3,3a, 4-tetrahydropyrrolo [1,2-a ] quinoxalin-5 (1H) -yl) propionate

i. Adding methyl 3- (7-formyl-8-methoxy-2, 3,3a, 4-tetrahydropyrrolo [1,2a ] quinoxaline-5 (1H) -yl) propionate and malononitrile into a proper amount of ethanol, adding a proper amount of piperidine, reacting at room temperature overnight, filtering, and drying a solid in vacuum to obtain the bifunctional fluorescent probe methyl 3- (7- (2, 2-dicyanovinyl) -8-methoxy-2, 3,3a, 4-tetrahydropyrrolo [1,2-a ] quinoxaline-5 (1H) -yl) propionate disclosed in claim 1.

3. The synthesis of bifunctional fluorescent probes as in claim 2, characterized in that the molar ratio of methyl 3- (7-formyl-8-methoxy-2, 3,3a, 4-tetrahydropyrrolo [1,2a ] quinoxalin-5 (1H) -yl) propionate to malononitrile in step i is 1: 1.2.

4. The use of a bifunctional fluorescent probe according to claim 1 for preparing a device for quantifying hydroxyl radicals and viscosity in an environment and simultaneously differentiating fluorescence imaging hydroxyl radicals and viscosity in living cells.

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